We have found that replication of HIV-1 in M. nemestrina is more vigorous in neonates than in adult animals. We have therefore initiated an experiment in which HIV-1 is passaged in M. nemestrina neonates with the expectation that viral virulence will increase in passage. The virus used for the passage experiment was isolated at 14 weeks postinfection from a neonate that was infected with a mixture of HIV-1lai and HIV-1NL4-3. This virus was inoculated into two neonates, and then, at 20 weeks postinfection, virus was isolated from these two animals to inoculate two more neonatal macaques. Immunological and virological data indicate that viral replication became more virulent during passage, but that so far the animals suffer no ill consequences from infection. The virus obtained after the first passage was also shown to replicate to higher titers in vitro in M. nemestrina cells, suggesting molecular adaptation. In an attempt to elucidate blocks for HIV-1 replication i n M. nemestrina cells we have established a system in which all the viral replication steps can be assessed quantitatively. We found that HIV-1 replication is blocked at the level of viral entry and at a later, postnuclear translocation, step. These data help us to design a molecularly engineered HIV-1 with enhanced replication characteristics in M. nemestrina cells. FUNDING NIH grant RR00166. Kimball, L.E. and Bosch, M.L. In vitro HIV-1 infection in Macaca nemestrina PBMCs is blocked at a step beyond reverse transcription. J. Med. Primatol. 27:99-103, 1998.